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41.

Background

Much progress has been made in understanding the 3D structure of proteins using methods such as NMR and X-ray crystallography. The resulting 3D structures are extremely informative, but do not always reveal which sites and residues within the structure are of special importance. Recently, there are indications that multiple-residue, sub-domain structural relationships within the larger 3D consensus structure of a protein can be inferred from the analysis of the multiple sequence alignment data of a protein family. These intra-dependent clusters of associated sites are used to indicate hierarchical inter-residue relationships within the 3D structure. To reveal the patterns of associations among individual amino acids or sub-domain components within the structure, we apply a k-modes attribute (aligned site) clustering algorithm to the ubiquitin and transthyretin families in order to discover associations among groups of sites within the multiple sequence alignment. We then observe what these associations imply within the 3D structure of these two protein families.

Results

The k-modes site clustering algorithm we developed maximizes the intra-group interdependencies based on a normalized mutual information measure. The clusters formed correspond to sub-structural components or binding and interface locations. Applying this data-directed method to the ubiquitin and transthyretin protein family multiple sequence alignments as a test bed, we located numerous interesting associations of interdependent sites. These clusters were then arranged into cluster tree diagrams which revealed four structural sub-domains within the single domain structure of ubiquitin and a single large sub-domain within transthyretin associated with the interface among transthyretin monomers. In addition, several clusters of mutually interdependent sites were discovered for each protein family, each of which appear to play an important role in the molecular structure and/or function.

Conclusions

Our results demonstrate that the method we present here using a k- modes site clustering algorithm based on interdependency evaluation among sites obtained from a sequence alignment of homologous proteins can provide significant insights into the complex, hierarchical inter-residue structural relationships within the 3D structure of a protein family.
  相似文献   
42.
Phosphorylation of proteins is an important mechanism used to regulate most cellular processes. Recently, we completed an extensive phosphoproteomic analysis of the core proteins that constitute the Saccharomyces cerevisiae centrosome. Here, we present a study of phosphorylation sites found on the mitotic exit network (MEN) proteins, most of which are associated with the cytoplasmic face of the centrosome. We identified 55 sites on Bfa1, Cdc5, Cdc14 and Cdc15. Eight sites lie in cyclin-dependent kinase motifs (Cdk, S/T-P), and 22 sites are completely conserved within fungi. More than half of the sites were found in centrosomes from mitotic cells, possibly in preparation for their roles in mitotic exit. Finally, we report phosphorylation site information for other important cell cycle and regulatory proteins.Key words: in vivo phosphorylation, yeast centrosome, mitotic exit network (MEN), cell cycle, protein kinase, Cdk (cyclin-dependent kinase)/Cdc28, Plk1 (polo-like kinase)/Cdc5Reversible protein phosphorylation leads to changes in targeting, structure and stability of proteins and is used widely to modulate biochemical reactions in the cell. We are interested in phosphoregulation of centrosome duplication and function in the yeast Saccharomyces cerevisiae. Centrosomes nucleate microtubules and, upon duplication during the cell cycle, form the two poles of the bipolar mitotic spindle used to segregate replicated chromosomes into the two daughter cells. Timing and spatial cues are highly regulated to ensure that elongation of the mitotic spindle and separation of sister chromatids occur prior to progression into late telophase and initiation of mitotic exit. The mitotic exit network (MEN) regulates this timing through a complex signaling cascade activated at the centrosome that triggers the end of mitosis, ultimately through mitotic cyclin-dependent kinase (Cdk) inactivation (reviewed in ref. 1).The major components of the MEN pathway (Fig. 1) are a Ras-like GTPase (Tem1), an activator (Lte1) with homology to nucleotide exchange factors, a GTPase-activating protein (GAP) complex (Bfa1/Bub2), several protein kinases [Cdc5 (Plk1 in humans), Cdc15 and Dbf2/Mob1] and Cdc14 phosphatase (reviewed in ref. 25). Tem1 initiates the signal for the MEN pathway when switched to a GTP-active state. Prior to activation at anaphase, it is held at the centrosome in an inactive GDP-bound state by an inhibiting GAP complex, Bfa1/Bub2.6 The Bfa1/Bub2 complex and the inactive Tem1 are localized at the mother centrosome destined to move into the budded cell upon chromosome segregation, whereas the activator Lte1 is localized at the tip of the budded cell. These separate localizations ensure that Lte1 and Tem1 only interact in late anaphase, when the mitotic spindle elongates.7,8 Lte1 has been thought to activate Tem1 as a nucleotide exchange factor, although more recent evidence suggests that it may instead affect Bfa1 localization.9 In addition, full activation of Tem1 is achieved through Cdc5 phosphorylation of the negative regulator Bfa1 10 and potentially through phosphorylation of Lte1. GTP-bound Tem1 is then able to recruit Cdc15 to the centrosome, allowing for Dbf2 activation.3 The final step in the MEN pathway is release of Cdc14 from the nucleolus, which is at least partially due to phosphorylation by Dbf211 an leads to mitotic cyclin degradation and inactivation of the mitotic kinase.2Open in a separate windowFigure 1Schematic representation of the MEN proteins and pathway. MEN protein localization is shown within a metaphase cell when mitotic exit is inhibited and in a late anaphase cell when mitotic exit is initiated. Primary inhibition and activation events are described below the cells.Recently, we performed a large-scale analysis of phosphorylation sites on the 18 core yeast centrosomal proteins present in enriched centrosomal preparations.12 In total, we mapped 297 sites on 17 of the 18 proteins and described their cell cycle regulation, levels of conservation and demonstrated defects in centrosome assembly and function resulting from mutating selected sites. MEN proteins were also identified in the centrosome preparations. This was expected, because Nud1, one of the 18 core centrosome components, is known to recruit several MEN proteins to the centrosome13 as part of its function in mitotic exit.14,15 As phosphorylation is essential to several steps in the MEN pathway, beginning with recruitment of Bfa1/Bub2 by phosphorylated Nud1,15 we were interested in mapping in vivo phosphorylation sites on the MEN proteins associated with centrosomes and identifying when they occur during the cell cycle.We combined centrosome enrichment with mass spectrometry analysis to examine phosphorylation from asynchronously growing cells.12 Centrosomes were also prepared from cells arrested in G1 and mitosis12 to monitor potentially cell cycle-regulated sites. We obtained significant coverage of a number of the MEN proteins, several of which have human homologs (and33, column 1), of which eight sites lie within Cdk/Cdc28 motifs [S/T(P)], (23 Mob1 and Dbf2 are known phosphoproteins24 for which we observed peptide coverage but no phosphorylation. Surprisingly, we did not detect phosphorylation on Bub2 despite the high peptide coverage; it is possible that the mitotic centrosome preparations (using a Cdc20 depletion protocol) affect the phosphorylation state of Bub2, as Bub2 is required for mitotic exit arrest in cdc20 mutants.25 Additionally, specific phosphorylation sites have not been mapped on Bub2, suggesting that modifications on this protein may be difficult to observe by mass spectrometry. Lte1 does not localize to the centrosome, and we did not recover Lte1 peptides in our preparations. Many phosphorylation events on MEN proteins were observed in mitotic centrosomal preparations, most likely in preparation for their subsequent role in exit from mitosis (
MEN ProteinSequence CoverageTotal SitesS/T (P) SitesHuman Homologs
Bfa198%352N/A
Cdc1480%102CDC14A, 14B2
Cdc1512%31MST1, STK4
Cdc541%73PLK1, PLK2, PLK3
Bub267%--N/A
Tem118%--RAB22, RAB22A
Mob113%--MOB1B, 1A, 2A, 2B
Dbf22%--STK38, LATS1
TOTAL558
Open in a separate window

Table 2

Cell cycle regulators of MEN proteins
Cell Cycle Regulator
CdkCdc5Cdc14Dbf2
Bfa16,10,23,2425
Cdc14212611
Cdc521,27
Cdc15282831
Open in a separate window

Table 3

All phosphorylation sites identified in MEN proteins Bfa1, Cdc14, Cdc15 and Cdc5
Open in a separate window
Open in a separate window
Open in a separate windowConservation of domains or of individual residues of proteins is often correlated with function.26 We utilized a protein fungal alignment tool (SGD: www.yeastgenome.org/) to analyze the conservation of the individual phosphorylated residues among selected Saccharomyces strains. If an amino acid substitution occurred, we noted whether the alternate residue could also be phosphorylated [serine (S) or threonine (T)], or whether it mimicked phosphorylation with a negative charge [aspartic (D) or glutamic (E) acid]. Using these criteria with the 55 phosphorylation sites, we found 22 that were completely identical among the fungi, two that were conserved as potential phosphorylation sites (6 Interestingly, Cdc5-T238 is also conserved in human polo-like kinases (Plk1–3). In another study, Mohl et al. tested nonphosphorylatable mutations of Dbf2 kinase motifs adjacent to the nuclear localization domain within Cdc14 phosphatase. One mutant allele of CDC14 wherein four Dbf2 motif sites were changed to alanines, includes our mapped site, S546 (20 While exceptionally rich clusters of phosphorylation sites (≥ 5/50 residues) are rare in the yeast proteome,27 the dense negative charge associated with phosphorylation clusters can enhance the rapidity and magnitude of the resulting cellular event. Two of the MEN proteins examined, Bfa1 (24 out of 35 total sites) and Cdc14 (5 out of 10 total sites), showed evidence of phosphorylation clustering (Fig. 2). Mutating groups of these clustered sites could provide insight into how the negatively charged regions affect protein localization and/or function.Open in a separate windowFigure 2Clustering of phosphorylation sites within the MEN proteins, Bfa1 and Cdc14. All phosphorylation sites within Bfa1 and Cdc14 are shown along the X-axis, representing the primary protein sequence and the Y-axis denoting the number of sites. Sites are considered clustered if there are at least 5 sites with a density ≥ 1 per 10 amino acids, and are marked with a horizontal bracket.In addition to proteins known to be associated with the yeast centrosome, such as the MEN proteins described, we recovered limited peptides from a number of other cell cycle and regulatory proteins. The high sensitivity with which mass spectrometry can detect modifications on proteins enabled the identification of in vivo phosphorylation sites that are cataloged in Open in a separate windowOpen in a separate windowOur large-scale centrosome enrichment and phosphorylation analysis has yielded a rich library of phosphorylation events on core centrosomal components, those involved in the mitotic exit network and additional regulatory proteins. Information regarding the phosphorylation state of various proteins throughout the cell will be useful in studying their control and function.?

Table 4

Summary of phosphorylation sites identified in centrosomes from different cell cycle stages and their conservation
Open in a separate window
Open in a separate window  相似文献   
43.
Protective mAbs and Cross-Reactive mAbs Raised by Immunization with Engineered Marburg Virus GPs     
Marnie L. Fusco  Takao Hashiguchi  Robyn Cassan  Julia E. Biggins  Charles D. Murin  Kelly L. Warfield  Sheng Li  Frederick W. Holtsberg  Sergey Shulenin  Hong Vu  Gene G. Olinger  Do H. Kim  Kevin J. Whaley  Larry Zeitlin  Andrew B. Ward  Cory Nykiforuk  M. Javad Aman  Jody Berry  Erica Ollmann Saphire 《PLoS pathogens》2015,11(6)
The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs) have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV) strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston), but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel “wing” feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV.  相似文献   
44.
An easy and reliable method for establishment and maintenance of leaf and root cell cultures ofArabidopsis thaliana     
CL Encina  M Constantin  J Botella 《Plant Molecular Biology Reporter》2001,19(3):245-248
Cell suspension cultures are useful for a wide range of biochemical and physiological studies, yet their production can be technically demanding and often unreliable. Here we describe a protocol for producing Arabidopsis cell suspension cultures that is reliable and easy to use.  相似文献   
45.
Expression and properties of diacylglycerol acyltransferase from cell-suspension cultures of oilseed rape     
Weselake RJ  Nykiforuk CL  Laroche A  Patterson NA  Wiehler WB  Szarka SJ  Moloney MM  Tari LW  Derekh U 《Biochemical Society transactions》2000,28(6):684-686
  相似文献   
46.
Changing concepts in plant hormone action   总被引:4,自引:0,他引:4  
Th.?GasparEmail author  C.?Kevers  O.?Faivre-Rampant  M.?Crèvecoeur  CL.?Penel  H.?Greppin  J.?Dommes 《In vitro cellular & developmental biology. Plant》2003,39(2):85-106
Summary A plant hormone is not, in the classic animal sense, a chemical synthesized in one organ, transported to a second organ to exert a chemical action to control a physiological event. Any phytohormone can be synthesized everywhere and can influence different growth and development processes at different places. The concept of physiological activity under hormonal control cannot be dissociated from changes in concentrations at the site of action, from spatial differences and changes in the tissue's sensitivity to the compound, from its transport and its metabolism, from balances and interactions with the other phytohormones, or in their metabolic relationships, and in their signaling pathways as well. Secondary messengers are also involved. Hormonal involvement in physiological processes can appear through several distinct manifestations (as environmental sensors, homeostatic regulators and spatio-temporal synchronizers, resource allocators, biotime adjusters, etc.), dependent on or integrated with the primary biochemical pathways. The time has also passed for the hypothesized ‘specific’ developmental hormones, rhizocaline, canlocaline, and florigen: root, stem, and flower formation result from a sequential control of specific events at the right places through a coordinated control by electrical signals, the known phytohormones and nonspecific molecules of primary and secondary metabolism, and involve both cytoplasmic and apoplastic compartments. These contemporary views are examined in this review.  相似文献   
47.
The variability of the hepatitis B virus genome: statistical analysis and biological implications     
Lauder  IJ; Lin  HJ; Lau  JY; Siu  TS; Lai  CL 《Molecular biology and evolution》1993,10(2):457-470
A statistical analysis of the nucleotide sequence variability in 14 published hepatitis B virus (HBV) genomes was carried out using parametric and nonparametric methods. A parametric statistical model revealed that the different regions of the genome differed significantly in their variability. The conclusion was supported by a nonparametric kernel-density model of the HBV genome. Genes S, C, and P, region X, the precore region, and the pre-S2/pre-S1 regions were ranked in order of increasing variability. In many instances, conserved regions of the genome identified with sequences of known function in HBV biology. However, other characterized regions (such as pre-S) showed much variability despite the involvement of their encoded peptides in specific functions. Point mutations that may result in the formation of stop codons and amino acid changes may affect the clinical picture of HBV infection and may be reflected in atypical serological patterns.   相似文献   
48.
Sequence, organization, and evolution of the A+T region of Drosophila melanogaster mitochondrial DNA   总被引:2,自引:0,他引:2  
Lewis  DL; Farr  CL; Farquhar  AL; Kaguni  LS 《Molecular biology and evolution》1994,11(3):523-538
The long (4.6-kb) A+T region of Drosophila melanogaster mitochondrial DNA has been cloned and sequenced. The A+T region is organized in two large arrays of tandemly repeated DNA sequence elements, with nonrepetitive intervening and flanking sequences comprising only 22% of its length. The first repeat array consists of five repeats of 338-373 bp. The second consists of four intact 464-bp repeats and a fifth partial repeat of 137 bp. Three DNA sequence elements are found to be highly conserved in D. melanogaster and in several Drosophila species with short A+T regions. These include a 300-bp DNA sequence element that overlaps the DNA replication origin and two thymidylate stretches identified on opposite DNA strands. We conclude that the length heterogeneity observed in the A+T regulatory region in mitochondrial DNAs from the genus Drosophila results from the expansion (and contraction) of the number of repeated DNA sequence elements. We also propose that the 300-bp conserved DNA sequence element, in conjunction with another primary sequence determinant, perhaps the adjacent thymidylate stretch, functions in the regulation of mitochondrial DNA replication.   相似文献   
49.
A qualitative investigation of smoke-free policies on hospital property     
Schultz AS  Finegan B  Nykiforuk CI  Kvern MA 《CMAJ》2011,183(18):E1334-E1344

Background:

Many hospitals have adopted smoke-free policies on their property. We examined the consequences of such polices at two Canadian tertiary acute-care hospitals.

Methods:

We conducted a qualitative study using ethnographic techniques over a six-month period. Participants (n = 186) shared their perspectives on and experiences with tobacco dependence and managing the use of tobacco, as well as their impressions of the smoke-free policy. We interviewed inpatients individually from eight wards (n = 82), key policy-makers (n = 9) and support staff (n = 14) and held 16 focus groups with health care providers and ward staff (n = 81). We also reviewed ward documents relating to tobacco dependence and looked at smoking-related activities on hospital property.

Results:

Noncompliance with the policy and exposure to secondhand smoke were ongoing concerns. Peoples’ impressions of the use of tobacco varied, including divergent opinions as to whether such use was a bad habit or an addiction. Treatment for tobacco dependence and the management of symptoms of withdrawal were offered inconsistently. Participants voiced concerns over patient safety and leaving the ward to smoke.

Interpretation:

Policies mandating smoke-free hospital property have important consequences beyond noncompliance, including concerns over patient safety and disruptions to care. Without adequately available and accessible support for withdrawal from tobacco, patients will continue to face personal risk when they leave hospital property to smoke.Canadian cities and provinces have passed smoking bans with the goal of reducing people’s exposure to secondhand smoke in workplaces, public spaces and on the property adjacent to public buildings.1,2 In response, Canadian health authorities and hospitals began implementing policies mandating smoke-free hospital property, with the goals of reducing the exposure of workers, patients and visitors to tobacco smoke while delivering a public health message about the dangers of smoking.25 An additional anticipated outcome was the reduced use of tobacco among patients and staff. The impetuses for adopting smoke-free policies include public support for such legislation and the potential for litigation for exposure to second-hand smoke.2,4Tobacco use is a modifiable risk factor associated with a variety of cancers, cardiovascular diseases and respiratory conditions.611 Patients in hospital who use tobacco tend to have more surgical complications and exacerbations of acute and chronic health conditions than patients who do not use tobacco.611 Any policy aimed at reducing exposure to tobacco in hospitals is well supported by evidence, as is the integration of interventions targetting tobacco dependence.12 Unfortunately, most of the nearly five million Canadians who smoke will receive suboptimal treatment,13 as the routine provision of interventions for tobacco dependence in hospital settings is not a practice norm.1416 In smoke-free hospitals, two studies suggest minimal support is offered for withdrawal, 17,18 and one reports an increased use of nicotine-replacement therapy after the implementation of the smoke-free policy.19Assessments of the effectiveness of smoke-free policies for hospital property tend to focus on noncompliance and related issues of enforcement.17,20,21 Although evidence of noncompliance and litter on hospital property2,17,20 implies ongoing exposure to tobacco smoke, half of the participating hospital sites in one study reported less exposure to tobacco smoke within hospital buildings and on the property.18 In addition, there is evidence to suggest some decline in smoking among staff.18,19,21,22We sought to determine the consequences of policies mandating smoke-free hospital property in two Canadian acute-care hospitals by eliciting lived experiences of the people faced with enacting the policies: patients and health care providers. In addition, we elicited stories from hospital support staff and administrators regarding the policies.  相似文献   
50.
Genome-wide assessment of imprinted expression in human cells     
Morcos L  Ge B  Koka V  Lam KC  Pokholok DK  Gunderson KL  Montpetit A  Verlaan DJ  Pastinen T 《Genome biology》2011,12(3):R25
  相似文献   
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